Functional foods and beverages typically have a health-promoting and/or disease-preventing property beyond their basic supply of nutrients. Functional foods and beverages have been developed by adding to these foods and beverages nutritional supplements, such as vitamins, antioxidants, and other nutraceuticals, which are generally intended to supplement the diet such that it becomes more nutritionally complete, and serve to prevent diseases. Other additives such as aroma compounds, flavors, dyes, and the like, may also be incorporated into foods and beverages.
However, solutions aimed at incorporating nutraceuticals and other additives in foods and beverages, particularly clear ones, at the nano-scale, are still very scarce. Incorporating additives into foods and beverages is still a major challenge in creating advanced functional foods. The challenges are especially great in acidic food products (pH around 2.0-4.2), and in nonfat and low fat foods. The main difficulties are encountered in dispersing, solubilizing, stabilizing and protecting the additives which are often sensitive bioactive compounds.
Further challenges are encountered when trying to add mainly, but not only, water insoluble additives, without compromising sensory properties of the food product. Moreover, stability and preservation of bioactivity need to be maintained throughout product shelf-life.
Maintaining transparency of clear beverages during enrichment is especially challenging, as it further requires the nutraceutical-loaded carrier to be small enough so as not to scatter light and be detected by the naked eye. Furthermore, any matrix containing bioactive ingredients within food or drink needs to be made of food-grade, GRAS (generally recognized as safe) materials and it is desirable that these materials be easily digestible to promote bioavailability and functionality of the bioactive additives. Lastly, a major difficulty arises from the fact that consumers prefer the product to be composed of natural ingredients only. Existing solutions include Gum Arabic-based emulsions, lipid-based systems such as microemulsions, cubosomes and hexosomes, and reformed casein micelles. Gum Arabic (GA) has been extensively used as an emulsifier and stabilizer in beverages, mainly for emulsifying aroma oils in acid drinks. It has good emulsifying and stabilizing capacities, and is considered a natural encapsulator for water-insoluble compounds, such as flavors and nutraceuticals such as lycopene, linoleic acid, and conjugated linoleic acid. GA may provide a reasonable solution in some conditions, but it is very expensive. It is also less stable at low pH conditions (e.g., pH 2.5), than at higher pH values, and it is very variable in composition. Previous experiments performed by some of the inventors showed that GA is a poor barrier to oxygen. Thus nutraceticals sensitive to oxidative stress such omega 3 and 6 fatty acids, PUFA, and oils had a very poor stability when encapsulated in GA (Dror-Katz, MSc thesis, Biotechnology and Food Engineering Dept., Technion, Israel Institute of Technology, 1995)
Lipid-based cubosomes and hexosomes are designed to give solutions for neutral pH conditions, but they are still in research stages. Additionally, these nano-vehicles are relatively large (hundreds of nanometers), thus, their dispersions are not transparent.
Microemulsions prepared from combinations of low molecular weight surfactants (e.g., Brij 96v and Tween 60), limonene and ethanol are another possible solution. However, existing microemulsions are not made of “all natural ingredients”.
Certain casein micelles are known in the art. Casein, which accounts for about 80% of milk protein, is naturally organized in micelles. Casein micelles (CM) are designed by nature to efficiently concentrate, stabilize and transport essential nutrients, mainly calcium and protein, for the neonate.
Naturally occurring micelles are spherical colloids, 50-500 nm in diameter (average of 150 nm), made of the main four caseins: αs1-casein (αs1-CN), αs2-CN, β-CN, and κ-CN (molar ratio ˜4:1:4:1 respectively). The caseins are held together in the micelle by hydrophobic interactions and by bridging of calcium-phosphate nanoclusters bound to serine-phosphate residues present within the casein molecules.
The structure of the casein micelles is important for their biological activity, for their stability and for providing good digestibility of the nutrients comprising the micelles. Harnessing the remarkable casein micelles natural nano-capsules for nano-encapsulation and stabilization of hydrophobic nutraceutical substances was suggested in the prior art. Semo et al., referred to the incorporation of such CM nano-capsules in dairy products without modifying their sensory properties (Semo E. Food Hydrocolloids 2007, 21; 936-42, and publication WO/2007/122613) and further suggested their use as delivery agents of sensitive health-promoting substances using natural GRAS (generally regarded as safe) ingredients.
U.S. Pat. No. 6,652,875 provides a formulation for the delivery of bioactive agents to biological surfaces comprising at least one isolated and purified casein protein or salt thereof in water. That disclosure relates to particular isolated and purified casein phosphoproteins in the form of casein calcium phosphate complexes intended to remain on the surface of oral cavity tissues or the gastrointestinal tract. There is neither teaching, nor suggestion regarding formation of nanoparticles, nor introduction of the bioactive compounds into nanoparticles. Furthermore the micelles comprise a casein protein selected from alpha-casein, beta-casein, kappa-casein, and mixtures thereof. This disclosure emphasizes the presence of divalent and trivalent metal ions.
U.S. Patent Application Publication No. 2002/0054914 teaches a calcium phosphate/drug core with casein micelles reconstructed as aggregates around the cores, forming micellar structures, for the delivery of pharmaceutical agents. According to that disclosure, casein molecules are arranged, presumably as micelles, around calcium phosphate particles containing the active drug, and are linked to the therapeutic agent-containing microparticles by mainly calcium phosphate and electrostatic bond interactions.
U.S. Patent Application Publication No. 2009/0029017 provides a protective system for lipids sensitive to oxidative processes by encapsulating them in a complex of casein and whey proteins. The emulsion is reported to stabilize the oxidizable lipid by decreasing its rate of oxidation. The emulsion is further reported to be heat stable which allows it to be heat treated and sterilized. However, the emulsion clearly requires a combination of both types of proteins; furthermore, the effect of low pH values and/or low temperature is not discussed. In fact, the pH is stated to be preferably between 6 and 9, with the upper end of the range even more preferred. Also the complex is stated to be formed by heating to between 70-100 degrees C.
Casein bound to a dextran copolymer nanoparticles encapsulating insoluble β-carotene were disclosed by Pan X. et al. (Journal of Colloid and Interface Science, 2007, 315; 456-63). The nanoparticles formed contained a casein and β-carotene core surrounded by a dextran shell. The particles were shown to have spherical shape and are stable in aqueous solution against dilution, pH change, ionic strength change, FeCl3 oxidation and long term storage. The casein-dextran nanoparticles were suggested as possible delivery agents for unstable and hydrophobic nutrients and drugs. However this disclosure requires the dextran copolymer for forming the nanoparticles.
In some cases caseinates have been used as microencapsulation wall materials. However, caseins forming such artificial capsules have lost the original micellar structure, and the generally larger size of caseinate microcapsules is more likely to impair product smoothness and clearness.
Casein micelles can be re-assembled in vitro, by simulating their formation in the Golgi system of the mammary gland. Reformed casein micelles may be used to encapsulate and protect hydrophobic nutraceuticals, however micelles are not reformed or able to remain stable at an acid pH.
U.S. Pat. No. 5,405,756 discloses acid soluble casein phosphopeptides prepared by enzymatic digestion of intact casein followed by step wise acidification of the digest causing precipitation of acid insoluble molecules. This procedure teaches that caseins tend to precipitate at pH values around the pI of the protein.
WO 2007/122613 described a system based on re-assembled casein micelles for the delivery of hydrophobic biologically active compounds in food and beverages. The invention relates specifically to the incorporation of such re-assembled casein micelles into low-fat or non-fat dairy products or other food or beverage products without adversely modifying their properties. The micelles of the invention are composed of sodium caseinate comprising at least the main four casein proteins and are re-assembled at neutral pH.
Thus, at present, there are only partial solutions to the problems listed above. There currently exists no solution having all of the desired attributes.